Preparation of bile acids and intermediates thereof

ABSTRACT

Synthetic methods for preparing deoxycholic acid and intermediates thereof are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/010,727, filed Jan. 20, 2011; which is a divisional of U.S. patentapplication Ser. No. 12/613,969, filed Nov. 6, 2009, issued as U.S. Pat.No. 7,994,351 on Aug. 9, 2011; which is a continuation of U.S. patentapplication Ser. No. 12/153,446, filed May 16, 2008, issued as U.S. Pat.No. 7,902,387 on; which claims the benefit under 35 U.S.C. 119(a) ofUnited Kingdom Application Serial No. 0807615.0, filed Apr. 25, 2008;each of which are hereby incorporated by reference into this applicationin their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the synthesis of deoxycholoic acid andpharmaceutically acceptable salts and intermediates thereof.

2. State of the Art

Rapid removal of body fat is an age-old ideal, and many substances havebeen claimed to accomplish such results, although few have shownresults. “Mesotherapy”, or the use of injectables for the removal offat, is not widely accepted among medical practitioners due to safetyand efficacy concerns, although homeopathic and cosmetic claims havebeen made since the 1950's. Mesotherapy was originally conceived inEurope as a method of utilizing cutaneous injections containing amixture of compounds for the treatment of local medical and cosmeticconditions. Although mesotherapy was traditionally employed for painrelief, its cosmetic applications, particularly fat and celluliteremoval, have recently received attention in the United States. One suchreported treatment for localized fat reduction, which was popularized inBrazil and uses injections of phosphatidylcholine, has been erroneouslyconsidered synonymous with mesotherapy. Despite its attraction as apurported “fat-dissolving” injection, the safety and efficacy of thesecosmetic treatments remain ambiguous to most patients and physicians.See, Rotunda, A. M. and M. Kolodney, Dermatologic Surgery 32: 465-480(2006) (“Mesotherapy and Phosphatidylcholine Injections: HistoricalClarification and Review”).

Recently published literature reports that the bile acid deoxycholicacid has fat removing properties when injected into fatty deposits invivo. See, WO 2005/117900 and WO 2005/112942, as well as US2005/0261258;US2005/0267080; US2006/127468; and US20060154906, all incorporatedherein by reference in their entirety including figures). Deoxycholateinjected into fat tissue has the effects of: 1) degrading fat cells viaa cytolytic mechanism; and 2) causing skin tightening. Both of theseeffects are required to mediate the desired aesthetic corrections (i.e.,body contouring). Because deoxycholate injected into fat is rapidlyinactivated by exposure to protein and then rapidly returns to theintestinal contents, its effects are spatially contained. As a result ofthis attenuation effect that confers clinical safety, fat removaltherapies typically require 4-6 sessions. This localized fat removalwithout the need for surgery is beneficial not only for therapeutictreatment relating to pathological localized fat deposits (e.g.,dyslipidemias incident to medical intervention in the treatment of HIV),but also for cosmetic fat removal without the attendant risk inherent insurgery (e.g., liposuction). See, Rotunda et al., Dermatol. Surgery 30:1001-1008 (2004) (“Detergent effects of sodium deoxycholate are a majorfeature of an injectable phosphatidylcholine formulation used forlocalized fat dissolution”) and Rotunda et al., J. Am. Acad. Dermatol.(2005: 973-978) (“Lipomas treated with subcutaneous deoxycholateinjections”), both incorporated herein by reference.

Pharmaceutical grade bile acid preparations are commercially availableat relatively low cost. This low cost is due to the fact that the bileacids are obtained from animal carcasses, particularly large animalssuch as cows and sheep. Importantly, as with all medicaments from animalsources, there is concern that the animal-derived bile acid products maycontain animal pathogens and other harmful agents such as animal ormicrobial metabolites and toxins, including bacterial toxins such aspyrogens.

Currently, the concerns regarding animal-derived products containinganimal pathogens and other harmful agents has been addressed by sourcingfrom isolated and inspected animals. For example, deoxycholic acid fromanimals in New Zealand are a source of bile acids for human use under USregulatory regimes, as long as the animals continue to remain isolatedand otherwise free of observable pathogens.

There remains a need for suitable quantities of efficacious bile acidssuch as deoxycholic acids that are known from the outset to be free frommoieties of animal origin (or pathogenic moieties capable of acting inan animal, particularly a mammal, and for human use, having adeleterious effect on a human), and other harmful agents such as animalor microbial metabolites, toxins, including bacterial toxins, such aspyrogens, for use as medicaments in humans.

SUMMARY OF THE INVENTION

The present invention provides methods and intermediates relating to thesynthesis of deoxycholic acid and pharmaceutically acceptable saltsthereof. The synthetically prepared deoxycholic acid can be used inadipolytic therapy for fat removal.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the similarity in dose-dependent decrease in cell survivalof primary human adipocytes upon treatment with synthetic sodiumdeoxycholic acid of the present invention in comparison tobovine-derived sodium deoxycholate (Sigma).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include singular and plural references unless the context clearlydictates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations. Each numerical parameter should atleast be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques.

As used herein, the term “comprising” is intended to mean that thecompounds and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the compounds or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompounds and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.Accordingly, it is intended that the methods and compounds can includeadditional steps and components (comprising) or alternatively includeadditional steps and compounds of no significance (consistingessentially of) or alternatively, intending only the stated methodssteps or compounds (consisting of).

The term “alkyl” refers to monovalent saturated aliphatic hydrocarbylgroups having from 1 to 10 carbon atoms and preferably 1 to 6 carbonatoms. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—),n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “oxidizing agent” refers to a reagent which can acceptelectrons in an oxidation-reduction reaction. In this way, oxygen can beadded to a molecule or hydrogen can be removed from a molecule.

The term “reducing agent” refers to a reagent which can donate electronsin an oxidation-reduction reaction, allowing hydrogen to be added to amolecule.

The term “acetylating reagent” refers to a reagent in which can add anacetyl (Ac) group CH₃C(O)— to an alcohol moiety of a molecule.

The term “acid” refers to regents capable of donating H⁺.

The term “Lewis acid” refers to an electron pair acceptor. Lewis acidsinclude oraganometallic reagents such as alkyl aluminum halides (e.g.Et₂AlCl and MeAlCl₂).

The term “hydrogenation conditions” refers to suitable conditions andcatalysts for introducing H₂ across one or more double bonds.Hydrogenation catalysts include those based on platinum group metals(platinum, palladium, rhodium, and ruthenium) such as Pd/C and PtO₂.

The term “olefination reagent” refers to regents that react with ketonesto form the corresponding olefins. The term “olefin forming conditions”refers to suitable conditions for carryout such transformations.Examples of such reagents include Wittig regeants and Wittig olefinationconditions.

The numbering of the steroidal scaffold as used herein follows thegeneral convention:

Accordingly, provided is a method for preparing deoxycholic acid (DCA)or a pharmaceutically acceptable salt thereof:

said method comprising

(a) reacting 9α-hydroxyandrost-4-en-3,17-dione 1.0 with H₂ underhydrogenation conditions to form compound 1.1

(b) reacting compound 1.1 with acid to form compound 1.2

(c) reacting compound 1.2 a reducing agent to form compound 1.3 amixture of 1.3 and 1.4

(d) reacting compound 1.3 with a two carbon olefination reagent underolefin forming conditions to form compound 1.5

(e) converting compound 1.5 to a compound of formula 1.6 wherein P is aprotecting group

(f) reacting a compound of formula 1.6 with an alkylpropiolateCH₂CH₂C(O)OR or an alkyl acrylate CH₂═CHC(O)OR wherein R is alkyl in thepresence of a Lewis acid to form a compound of formula 1.7 wherein P isa protecting group, R is a alkyl, and the dashed line

is a single or double bond;

(g) reacting a compound of formula 1.7 with H₂ under hydrogenationconditions to form a compound of formula 1.8 wherein P is a protectinggroup and R is alkyl

(h) reacting compound of formula 1.8 with an oxidizing agent to form acompound of formula 1.9 wherein P is a protecting group and R is alkyl

(i) reacting a compound of formula 1.9 with H₂ under hydrogenationconditions to form compound of formula 2.0 wherein P is a protectinggroup and R is alkyl

(j) reacting compound of formula 2.0 with a reducing agent to form acompound of formula 2.1 wherein P is a protecting group and R is alkyl

and

(k) exposing compound of formula 2.1 to deprotection and hydrolysisconditions to form deoxycholic acid.

The present invention also provides the following intermediates shown inScheme 1 below wherein Ac and R are as defined above.

In one embodiment, the hydrogenation conditions of part (a) comprises aPd/C catalyst.

In one embodiment, the acid of part (b) is a mineral acid. In someaspects, the mineral acid is H₂SO₄.

In one embodiment, the reducing agent of part (c) is LiAl(OtBu)₃H.

In one embodiment, the two carbon olefination reagent of part (d) is aWittig agent such as Ph₃PCH₂CH₃ ⁺Br⁻.

In one embodiment, the protecting group P of compound 1.6-2.1 is—C(O)CH₃. In some aspects compound 1.5 is exposed to acylationconditions to form 1.6a, such as by treatment of 1.5 with aceticanhydride and an organic base such as Et₃N, pyridine, and/ordimethylaminopyridine.

In one embodiment, the Lewis acid of part (f) is EtAlCl₂.

In one embodiment, the alkylpropiolate of part (f) is methylpropriolate.

In one embodiment, the alkyl acrylate of part (f) is methylacrylate.

In one embodiment, the hydrogenation conditions of part (g) comprises aPtO₂ or Pd/C catalyst.

In one embodiment, the oxidizing agent of part (h) is CrO₃.

In one embodiment, the hydrogenation conditions of part (i) comprises aPd/C catalyst.

In one embodiment, the reducing agent of part (j) is LiAl(OtBu)₃H.

In one embodiment, the deprotection and hydrolysis conditions of part(k) when P is —C(O)CH₃ comprises reacting compound 2.1a with an alkaliearth hydroxide, alkali earth alkoxide, or a mixture of both.

In one embodiment, the alkali earth alkoxide is LiOH.

In one embodiment, salts of deocycholoic acid can be prepared byreaction with an alkali earth metal alkoxide or hydroxide. Salts ofdeocycholoic acid include the sodium (Na⁺), potassium (K⁺), and lithium(Li⁺) salts.

In one embodiment, provided is an intermediate compound selected fromthe group consisting of

-   9α-Hydroxy-5β-androstan-3,17-dione (1.1);-   5β-Androst-9(11)-en-3,17-dione (1.2);-   (Z)-3α-Hydroxy-5β-pregna-9(11),17(20)-diene (1.5);-   (Z)-3α-Acetoxy-5β-pregna-9(11),17(20)-diene (1.6);-   (E)-Methyl 3α-acetoxy-5β-chol-9(11), 16,22-trien-24-oate (1.7a);-   Methyl 3α-acetoxy-5β-chol-9(11), 16-dien-24-oate (1.7b);-   Methyl 3α-hydroxy-5β-chol-9(11)-en-12-one-24-oate (1.9a); and-   Methyl 3α-acetoxy-5β-cholan-12-one-24-oate (2.0a).

The compounds of preferred embodiments can be prepared from readilyavailable starting materials using the following general methods andprocedures. It will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

The starting materials and reagents for the reactions described hereinare generally known compounds or can be prepared by known procedures orobvious modifications thereof. For example, many of the startingmaterials and reagents are available from commercial suppliers such asAldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif.,USA), Emka-Chem or Sigma (St. Louis, Mo., USA). Others may be preparedby procedures, or obvious modifications thereof, described in standardreference texts such as Fieser and Fieser's Reagents for OrganicSynthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry ofCarbon Compounds, Volumes 1-5 and Supplementals (Elsevier SciencePublishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons,1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4^(th)Edition), and Larock's Comprehensive Organic Transformations (VCHPublishers Inc., 1989).

The various starting materials, intermediates, and compounds of thepreferred embodiments may be isolated and purified where appropriateusing conventional techniques such as precipitation, filtration,crystallization, evaporation, distillation, and chromatography.Characterization of these compounds may be performed using conventionalmethods such as by melting point, mass spectrum, nuclear magneticresonance, and various other spectroscopic analyses.

The foregoing and other aspects of the embodiments disclosed herein maybe better understood in connection with the following examples.

EXAMPLES

In the examples below and elsewhere in the specification, the followingabbreviations have the indicated meanings. If an abbreviation is notdefined, it has its generally accepted meaning.

AcOH Acetic acid Ac₂O Acetic anhydride CrO₃ Chromium trioxide DCADeoxycholic acid DCM (CH₂Cl₂) Dichloromethane DMF N,N-DimethylformamideEtOAc Ethyl acetate EtAlCl₂ Ethyl aluminum dichloride Hz Hertz HPLC Highpressure liquid chromatography HCl Hydrochloric acid LiOH Lithiumhydroxide Na₂SO₄ Sodium sulfate MHz Megahertz min Minutes MeOH Methanolmmol millimole mL milliliter mol mole Obs Observed HClO₄ Perchloric acidPtO₂ Platinum oxide Pd/C Palladium on carbon H₂SO₄ Sulphuric acid DMAP4-Dimethylaminopyridine LiAl(O^(t)Bu)₃H Lithium tri-tert-butoxyaluminumhydride KBr Potassium bromide K—O^(t)Bu Potassium tert-butoxide RepReported NaOH Sodium hydroxide THF Tetrahydrofuran TEA Triethylamine TLCThin layer chromatography Wt Weight CONC Concentrated ACN AcetonitrileTFA Trifluoroacetic acid

General:

All manipulations of oxygen- and moisture-sensitive materials wereconducted with standard two-necked flame dried flasks under an argon ornitrogen atmosphere. Column chromatography was performed using silicagel (60-120 mesh). Analytical thin layer chromatography (TLC) wasperformed on Merck Kiesinger 60 F₂₅₄ (0.25 mm) plates. Visualization ofspots was either by UV light (254 nm) or by charring with a solution ofsulfuric acid (5%) and p-anisaldehyde (3%) in ethanol.

Apparatus:

Proton and carbon-13 nuclear magnetic resonance spectra (¹H NMR and ¹³CNMR) were recorded on a Varian Mercury-Gemini 200 (¹H NMR, 200 MHz; ¹³CNMR, 50 MHz) or a Varian Mercury-Inova 500 (¹H NMR, 500 MHz; ¹³C NMR,125 MHz) spectrometer with solvent resonances as the internal standards(¹H NMR, CHCl₃ at 7.26 ppm or DMSO at 2.5 ppm and DMSO-H₂O at 3.33 ppm;¹³C NMR, CDCl₃ at 77.0 ppm or DMSO at 39.5 ppm). ¹H NMR data arereported as follows: chemical shift (δ, ppm), multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, br=broad, m=multiplet), couplingconstants (Hz), and integration. Infrared spectra (FT-IR) were run on aJASCO-460′ model. Mass spectra were obtained with a Perkin ElmerAPI-2000 spectrometer using ES⁺ mode. Melting points were determinedusing a LAB-INDIA melting point measuring apparatus and are uncorrected.HPLC chromatograms were recorded using a SHIMADZU-2010 model with a PDAdetector. Specific optical rotations were determined employing aJASCO-1020 at 589 nm and are uncorrected.

Chemicals:

Unless otherwise noted, commercially available reagents were usedwithout purification. Diethyl ether and THF were distilled fromsodium/benzophenone. Laboratory grade anhydrous DMF, commerciallyavailable DCM, ethyl acetate and hexane were used.

Example 1 9α-Hydroxy-5β-androstan-3,17-dione (1.1)

To a solution of 9α-hydroxyandrost-4-en-3,17-dione 1.0 (30.0 g, 99.3mol) in DMF (150 mL) was added 10% of Pd/C (2.1 g) and the resultingslurry was hydrogenated in a Parr apparatus (60 psi) for 12 h. Uponcomplete disappearance of starting material, as evidenced by TLC, thecrude reaction mixture was filtered through a small pad of Celite®, andthe solvent was removed under vacuum to provide a colorless solid (30.0g). This solid was combined with acetone (90 mL.) at 0° C. and theresulting slurry was stirred for 1 h. It was then filtered, washed withchilled (0° C.) acetone (30 mL) and dried under vacuum in the samefiltration funnel at room temperature to afford compound 1.1 (26.0 g,86%).

TLC: p-anisaldehyde charring, R_(f) for 1.1=0.48 and R_(f) for 1.0=0.30.

TLC mobile phase: 30%-EtOAc in DCM.

¹H NMR (500 MHz, CDCl₃): δ=2.37-2.40 (m, 1H), 2.02-.2.11 (m, 2H),1.31-1.91 (m, 19H), 0.96 (s, 3H), 0.84 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=221.0, 95.7, 80.1, 47.0, 43.6, 38.6, 38.5,37.1, 35.9, 33.41, 32.9, 32.0, 27.9, 26.9, 21.5, 20.2, 20.0, 12.6.

Mass (m/z)=305.0[M⁺+1], 322.0 [M⁺+18].

IR (KBr)=3443, 2938, 1722, 1449, 1331, 1138 cm⁻¹.

m.p=213-216° C. (from DMF and acetone)

[α]_(D)=+116 (c=1% in CHCl₃).

ELSD Purity: more than 99%, ret. time=8.15, 9-HAD ret. time=3.88,5α-isomer of Cmpd 121 ret. time=4.91 (Water symmetry 250×4.6 mm, 5 um,C18), Water:ACN (40:60).

Example 2 5β-Androst-9(11)-en-3,17-dione (1.2)

To a solution of compound 1.1 (26.0 g, 85.4 mmol) in DCM (520 mL) wasadded concentrated sulfuric acid (7.53 g, 76.8 mmol) over 15 minutesunder an inert atmosphere at 10° C. The temperature was raised to 25° C.and the resulting solution was stirred for 2 h. At this point no morestarting material remained as evidenced by TLC. The reaction wasquenched by the addition of 10% aqueous NaHCO₃ solution (200 mL). Thelayers were separated and the aqueous layer was extracted twice with DCM(2×100 mL). The organic layers were combined and washed sequentiallywith water (100 mL) and saturated brine solution (100 mL). The organicphase was then dried over Na₂SO₄ (75 g) and filtered. The filtrate wasevaporated under vacuum to provide compound 1.2 (23.0 g, 94%) as anoff-white solid. This product was used “as is” in the next step withoutfurther purification.

TLC: p-anisaldehyde charring, R_(f) for 1.2=0.76 and R_(f) for 1.1=0.44.

TLC mobile phase: 30%-EtOAc in DCM.

¹H NMR (500 MHz, CDCl₃): δ=5.61 (s, 1H), 2.47-2.57 (m, 2H), 2.24-2.42(m, 4H), 2.05-2.20 (m, 3H), 1.86-1.99 (m, 2H), 1.84-1.85 (d, J=6 Hz 1H),1.57-1.63 (m, 5H), 1.37-1.40 (d, J=13.5 Hz, 1H) 1.25-1.28 (dd, J=4.0,13.5 Hz, 1H), 1.17 (s, 3H) 0.85 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=221.3, 212.8, 140.1, 118.5, 48.5, 45.9,44.3, 43.5, 39.0, 38.0, 37.3, 36.1, 35.8, 33.3, 28.8, 26.0, 25.5, 22.5,13.9.

Mass (m/z)=287 [M⁺+1], 304 [M⁺+18].

IR (KBr)=3450, 2913, 1737, 1707, 1413, 1403, 1207 cm⁻¹.

m.p.=143.4-145.9° C. (from DCM)

[α]_(D)=+142 (c=1% in CHCl₃).

ELSD Purity: 99.7%, Retention time=5.04, (Inertsil ODS 3V 250×4.6 mm, 5um), ACN: 0.1%

TFA in water (90:10).

Example 3 3α-Hydroxy-5β-androst-9(11)-en-17-one (1.3)

A THF solution of lithium tri-tert-butoxyaluminum hydride (1.0 M, 84.4mL, 84.4 mmol) was added to a cold (−40° C.) solution of compound 1.2(23.0 g, 80.4 mmol) in THF (230 mL) under an inert atmosphere. Theresulting reaction mixture was stirred for 2 h. At this point thereaction was determined to be complete, as evidenced by TLC, and thereaction mixture was quenched by adding a mixture of 1N HCl (200 mL) andethyl acetate (230 mL). The resulting two phase mixture was separatedand the aqueous layer was extracted twice with ethyl acetate (2×100 mL).The organic phases were combined and washed sequentially with water (150mL) and saturated brine solution (100 mL). The organic phase was thendried over Na₂SO₄ (75 g) and filtered. The filtrate was evaporated undervacuum to afford compound 1.3 (23.0 g) as an off-white solid. The abovecrude product was used “as is” in the next step without purification.

TLC: p-anisaldehyde charring, R_(f) for 1.3=0.44 and R_(f) for 1.2=0.74.

TLC mobile phase: 30%-EtOAc in DCM (30%).

¹H NMR (500 MHz, CDCl₃): δ=5.41-5.42 (d, J=6.0 Hz, 1H), 3.65-3.66 (m,1H), 2.43-2.48 (m, 1H), 1.98-2.18 (m, 6H), 1.74 (s, 2H), 1.48-1.56 (m,5H), 1.377-1.45 (m, 3H), 1.18-1.28 (m, 3H), 1.08 (s, 3H), 0.80 (s, 3H)

¹³C NMR (125 MHz, CDCl₃): δ=222.0, 140.9, 118.3, 71.9, 48.6, 45.9, 41.7,38.8, 37.8, 36.2, 36.0, 35.7, 33.4, 31.7, 29.5, 26.5, 26.0, 22.7, 13.9.

Mass (m/z)=289.0 [M⁺+1], 306.0 [M⁺+18].

IR (KBr)=3463, 2960, 2871, 1729, 1366, 1165, 1084, 1041 cm⁻¹.

m.p.=165-167.5° C. (EtOAc/hexanes mixture).

[α]_(D)=+161 (c=1% in CHCl₃).

ELSD Purity: ˜93%, Retention time=5.23, (Inertsil ODS 3V 250×4.6 mm, 5um), ACN: 0.1% TFA in water (90:10).

Example 4 (Z)-3α-Hydroxy-5β-pregna-9(11),17(20)-diene (1.5)

A solution of potassium tert-butoxide in THF (1M, 230 mL, 231 mmol) wasadded drop wise to a suspension of ethyltriphenylphosphonium bromide(88.7 g, 239 mmol) in THF (150 mL) over 1 h at 25° C. The resulting darkred colored mixture was stirred for an additional 1 h at 25° C. Asolution of compound 1.3 (23.0 g, 79.7 mmol) in THF (230 mL) was addedslowly to the red-colored mixture at 25° C. The resulting mixture wasstirred for 3-4 h, at which point it was determined to be complete byTLC. The reaction was quenched by adding saturated aqueous NH₄Clsolution (75 mL). The phases were separated and the aqueous layer wasextracted three times with EtOAc (3×150 mL). The organic fractions werecombined, washed with saturated brine solution (100 mL), dried overNa₂SO₄ (75 g), and filtered. The filtrate was concentrated under vacuumand the crude solid was purified by column chromatography [49 mm (W)×600mm (L), 60-120 mesh silica, 300 g] eluting with ethyl acetate/hexanes(1:9). The fractions containing product were combined and concentrated,providing compound 1.5 (19.1 g, 80.0%) as a white solid.

TLC: p-anisaldehyde charring, R_(f) for 1.5=0.72 and R_(f) for 1.3=0.46.

TLC mobile phase: 30%-EtOAc in DCM.

¹H NMR (500 MHz, CDCl₃): δ=5.38 (s, 1H), 5.18-5.19 (d, J=6.5 Hz 1H),3.62-3.66 (m, 1H), 2.35-2.38 (d, J=15 Hz, 3H), 2.23-2.25 (m, 1H),1.97-2.07 (m, 3H), 1.64-1.75 (m, 6H), 1.32-1.55 (m, 6H), 1.17-1.24 (m,4H), 1.06 (s, 3H), 0.79 (s, 3H).

¹³CNMR (125 MHz, CDCl₃): δ=150.1, 140.6, 119.6, 114.2, 72.2, 53.6, 42.0,41.9, 39.6, 38.6, 37.9, 35.7, 35.6, 31.9, 31.8, 29.5, 26.9, 26.8, 25.5,16.9, 13.3.

Mass (m/z)=301[M⁺+1], 318[M⁺+18].

IR (CHCl₃)=3304, 3033, 2925, 2863, 1449, 1368, 1040, 823 cm⁻¹.

mp=146-147.3° C. (EtOAc/hexanes mixture).

[α]_(D)=+84.4 (c=1% in CHCl₃).

ELSD Purity: 99.8%, Retention time=16.07, (Inertsil ODS 3V 250×4.6 mm, 5um), ACN: 0.1% TFA in water (90:10).

Example 5 (Z)-3α-Acetoxy-5β-pregna-9(11),17(20)-diene (1.6a)

Compound 1.5 (19.0 g, 63 mmol) was dissolved in CH₂Cl₂ (380 mL).Triethylamine (17.6 mL, 126.6 mmol), DMAP (0.772 g, 6 mmol) and aceticanhydride (8.98 mL, 94 mmol) were added sequentially at 25° C. under anitrogen atmosphere. The resulting solution was stirred for 2 h at 25°C., at which point the reaction was determined by TLC to be complete.The reaction was quenched by the addition of ice-water (100 mL) and thephases were separated. The aqueous layer was extracted three times withDCM (3×150 mL). The organic fractions were combined and washed withsaturated brine solution (100 mL), dried over anhydrous Na₂SO₄ (50 g),and filtered. The filtrate was concentrated under vacuum to affordcompound 1.6a (22.0 g, 95% yield) as an off-white solid.

TLC: p-anisaldehyde charring, R_(f) for 1.6=0.5 and R_(f) for 1.5=0.15.

TLC mobile phase: 10%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=5.38 (s, 1H), 5.18-5.20 (d, J=6.5 Hz, 1H),4.72-4.76 (m, 1H), 2.35-2.40 (m, 3H), 2.22-2.25 (m, 1H), 2.03-2.09 (m,3H), 2.01 (s, 3H), 1.49-1.98 (m, 10H), 1.31-1.41 (m, 2H), 1.16-1.27 (m,3H), 1.07 (s, 3H), 0.79 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=170.5, 150.0, 140.4, 119.6, 114.3, 74.7,53.5, 42.0, 41.7, 39.6, 38.6, 35.6, 35.3, 33.8, 31.9, 29.5, 27.8, 26.7,26.6, 25.5, 21.3, 16.9, 13.2

Mass (m/z)=342.9 [M⁺+1], 360 [M⁺+18].

IR (CHCl₃)=3440, 3035, 1730, 1451, 1367, 1258, 1028 cm⁻¹.

mp=93.9-97.8° C. (EtOAc/hexanes mixture)

[α]_(D)=+109 (c=1% in CHCl₃).

HPLC purity: 97.62%; Retention time=17.7, (Zorbax SB, C18; 250×4.6 mm, 5um), ACN: 0.1% TFA in water (90:10).

Example 6 (E)-Methyl 3α-acetoxy-5β-chol-9(11), 16,22-trien-24-oate(1.7a)

Ethyl aluminum dichloride (104.5 mL, 192 mmol, 1.8 M in toluene) wasadded to a solution of methyl propiolate (13.58 mL, 153 mmol) in DCM(100 mL) at 0° C. under inert atmosphere. The resulting solution wasstirred for 15 min and then compound 1.6a (22 g, 64.3 mmol) was added.After stirring for an additional 20 min at 0° C., the temperature wasraised to 25° C. and held there for a further 18 h. At this point thereaction was determined to be complete by TLC, and the mixture waspoured into cold (0° C.) water (200 mL). The phases were separated andthe aqueous layer was extracted with DCM (150 mL). The organic layerswere combined and washed sequentially with water (200 mL) and saturatedbrine solution (100 mL). It was then dried over anhydrous Na₂SO₄ (40 g)and filtered. The filtrate was concentrated under vacuum and theresulting solid was purified by slurring in methanol (280 mL) to providecompound 1.7a (17.5 g 68%) as a white solid.

TLC: p-anisaldehyde charring, R_(f) for 1.7a=0.32 and R_(f) for1.6a=0.5.

TLC mobile phase: 10%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=6.92-6.926 (q, J=7.5, 15.5 Hz, 1H), 5.80-5.83(d, J=16 Hz, 1H), 5.37-5.43 (m, 2H), 4.73-4.75 (m, 1H), 3.73 (s, 3H),3.02-3.04 (t, J=6.5 Hz, 1H), 2.15-2.23 (m, 3H), 2.05-2.08 (m, 3H), 2.01(s, 3H), 1.48-1.99 (m, 8H), 1.24-1.34 (m, 2H), 1.20-1.21 (d, J=5 Hz,3H), 1.11-1.17 (m, 1H), 1.07 (s, 3H), 0.67 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=170.5, 167.2, 155.0, 153.7, 141.6, 124.0,118.8, 118.7, 74.6, 53.9, 51.3, 45.7, 41.7, 38.8, 37.1, 35.5, 35.3,34.6, 33.7, 31.8, 29.5, 27.7, 26.5, 26.5, 21.3, 19.7, 15.7.

Mass (m/z)=444.0 [M⁺+18].

IR (KBr)=3443, 3030, 2930, 1719, 1650, 1247, 1359, 1032, 1170 cm⁻¹.

m.p.=114-116° C. (from methanol)

[α]_(D)=+102 (c=1% in CHCl₃).

ELSD Purity: 99.7%, Retention time=19.57, (Inertsil ODS 3V 250×4.6 mm, 5um), ACN: 0.1% TFA in water (90:10).

Example 7 Methyl 3α-acetoxy-5β-chol-9(11)-en-24-oate (1.8a)

To a solution of compound 1.7a (17.5 g, 41 mmol) in EtOAc (350 mL) wasadded PtO₂ (4.37 g), and the resulting slurry was hydrogenated in a Parrapparatus (70 psi) for 14-16 h. At this point the reaction wasdetermined to be complete by TLC. The mixture was filtered through asmall plug of Celite® and the solvent was removed under vacuum,affording compound 1.8a (17.0 g, 96.0%) as a white solid. The aboveproduct was used in the next step without further purification.

TLC: p-anisaldehyde charring, R_(f) for 1.8a=0.32 and R_(f) for1.7a=0.30.

TLC mobile phase: 10%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=5.31 (s, 1H), 4.73 (m, 1H), 3.66 (s, 3H),2.03-2.37 (m, 7H), 2.01 (s, 3H), 1.09-1.98 (m, 18H), 1.06 (s, 3H),0.91-0.92 (d, J=6.0 Hz, 3H), 0.59 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=174.6, 170.5, 139.8, 119.5, 74.8, 56.0,53.3, 51.4, 41.9, 41.7, 40.9, 38.5, 36.4, 35.4, 35.2, 33.8, 31.0, 30.9,29.5, 28.2, 27.8, 26.8, 26.7, 25.2, 21.4, 17.9, 11.5

Mass (m/z)=448.2 [M⁺+18].

IR (KBr)=3435, 3039, 2941, 1729, 1448, 1435, 1252, 1022 cm⁻¹.

m.p.=122.1-123.9° C. (from EtOAc).

[α]_(D)=+56 (c=1% in CHCl₃)

ELSD Purity: 97.7%: Retention time=14.57 (ZORBAX SB C-18 150×4.6 mm, 5um, ACN: 0.1% TFA in water (90:10)

Example 8 Methyl 3α-acetoxy-5β-chol-9(11), 16-dien-24-oate (1.7b)

Ethyl aluminum dichloride (14.2 mL, 25 mmol, 1.8M in toluene) was addedto a solution of methyl acrylate (1.89 mL, 20 mmol) in DCM (60 mL) at 0°C. under inert atmosphere. The resulting solution was stirred for 15 minand then compound 1.6a (3 g, 8.7 mmol) was added. After stirring for anadditional 20 min at 0° C., the temperature was raised to 25° C. andheld there for a further 18 h. At this point the reaction was determinedto be complete by TLC, then the mixture was poured into cold (0° C.)water (60 mL). The phases were separated and the aqueous layer wasextracted with DCM (60 mL). The organic layers were combined and washedsequentially with water (50 mL) and saturated brine solution (100 mL).It was then dried over anhydrous Na₂SO₄ (5 g) and filtered. The filtratewas concentrated under vacuum, providing compound 1.7b (2.6 g, 70%).

TLC mobile phase: 10%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=5.34-5.43 (m, 2H), 4.73-4.75 (m, 1H), 3.73(s, 3H), 2.15-2.34 (m, 6H), 2.05-2.08 (m, 3H), 2.01 (s, 3H), 1.48-1.99(m, 9H), 1.24-1.34 (m, 3H), 1.20-1.21 (d, J=5 Hz, 3H), 1.11-1.17 (m,1H), 1.07 (s, 3H), 0.67 (s, 3H).

ELSD Purity: 93.9%, ret. time=4.55, (Water symmetry shield 250×4.6 mm5μ, ACN:100%

Example 9 Methyl 3α-acetoxy-5β-chol-9(11)-en-24-oate (1.8a)

To a solution of compound 1.7a (3 g, 7 mmol) in EtOAc (60 mL) was added10% Pd/C (300 mg, 10% wt/wt), and the resulting slurry was hydrogenatedin a Parr apparatus (70 psi) for 14-16 h. At this point the reaction wasdetermined to be complete by TLC (10% ethyl acetate in hexanes). Themixture was filtered through a small plug of Celite® and the solvent wasremoved under vacuum, affording compound 1.8a (2.6 g, 86%) as a whitesolid.

¹H NMR (500 MHz, CDCl₃): δ=5.31 (s, 1H), 4.73 (m, 1H), 3.66 (s, 3H),2.37-2.03 (m, 7H), 2.01 (s, 3H), 1.98-1.09 (m, 18H), 1.06 (s, 3H),0.92-0.91 (d, J=6.0 Hz, 3H), 0.59 (s, 3H).

ELSD Purity: 95.9%, ret. time=4.75, (Water symmetry shield 250×4.6 mm5μ, ACN:Water (60:40)

Example 10 Methyl 3α-hydroxy-5β-chol-9(11)-en-12-one-24-oate (1.9a)

CrO₃ (17.0 g, 170 mmol) was added to a solution of compound 1.8a (17 g,39.5 mmol) in AcOH (270 mL). The resulting mixture was heated at 50° C.for 24-36 h. Upon complete disappearance of the starting material byTLC, the solvent was evaporated under vacuum and the crude material wasdissolved in ethyl acetate (400 mL) and water (200 mL). The two phaseswere separated and the organic layer was washed twice with water (2×100mL) and then once with saturated brine solution (100 mL). The organicphase was dried over anhydrous Na₂SO₄ (40 g) and filtered. The filtratewas concentrated under vacuum and the resulting solid was purified bycolumn chromatography [49 mm (W)×600 mm (L), 60-120 mesh silica, 120 g],eluting with ethyl acetate/hexane (1:5) [10 mL fractions, 3 mL/minelution, monitored by TLC and detected with UV light (254 nm) lamp]. Theproduct-containing fractions were combined and concentrated under vacuumto afford compound 1.9a (8.8 g, 50% yield) as a white solid.

TLC: p-anisaldehyde charring, R_(f) for 1.9a=0.28 and R_(f) for18a=0.52.

TLC mobile phase: 20%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=5.71 (s, 1H), 4.71-4.75 (m, 1H), 3.66 (s,3H), 2.37-2.42 (m, 3H), 2.02-2.31 (m, 2H), 2.0 (s, 3H), 1.67-1.98 (m,9H), 1.24-1.56 (m, 9H), 1.19 (s, 3H), 1.01-1.02 (d, J=6.5 Hz, 3H), 0.90(s, 3H).

¹³C NMR (500 MHz, CDCl₃): δ=204.9, 174.5, 170.4, 163.8, 123.6, 73.7,53.4, 53.0, 51.3, 47.2, 41.7, 39.8, 37.7, 35.2, 35.0, 33.9, 31.4, 30.5,29.6, 27.6, 27.3, 26.4, 26.1, 24.1, 21.2, 19.4, 10.6.

Mass (m/z)=445.0 [M⁺+1], 462.0 [M⁺+18].

IR=3437, 3045, 2946, 2870, 1729, 1680, 1252, 1168, 1020, cm⁻¹.

m.p.=137-139° C. (from EtOAc/hexanes mixture).

[α]_(D)=+93 (c=1% in CHCl₃).

ELSD Purity: 94.6%: Retention time=8.68 (Inertsil ODS 3V, 250×4.6 mm, 5um, ACN:Water (60:40)

Example 11

Methyl 3α-acetoxy-5β-cholan-12-one-24-oate (2.0a)

10% Pd/C (900 mg) was added to a solution of compound 1.9a (2.0 g, 4.5mmol) in EtOAc (150 mL) and the resulting slurry was hydrogenated in aParr apparatus (50 psi) at 50° C. for 16 h. At this point the reactionwas determined to be complete by TLC. The mixture was filtered through asmall plug of Celite® and the solvent was removed under vacuum,providing compound 2.0a (1.6 g, 80% yield) as a white solid.

TLC: p-anisaldehyde charring, R_(f) for 2.0=0.36 and R_(f) for 1.9=0.32.

TLC mobile phase: 20%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=4.67-4.71 (m, 1H), 3.66 (s, 3H), 2.45-2.50(t, J=15 Hz, 2H), 2.22-2.40 (m, 1H), 2.01 (s, 3H), 1.69-1.96 (m, 9H),1.55 (s, 4H), 1.25-1.50 (m, 8H), 1.07-1.19 (m, 2H), 1.01 (s, 6H),0.84-0.85 (d, J=7.0 Hz, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=214.4, 174.5, 170.4, 73.6, 58.5, 57.4, 51.3,46.4, 43.9, 41.2, 38.0, 35.6, 35.5, 35.2, 34.8, 32.0, 31.2, 30.4, 27.4,26.8, 26.2, 25.9, 24.2, 22.6, 21.2, 18.5, 11.6,

Mass (m/z)=447.0 [M⁺+1], 464.0 [M⁺+18].

IR (KBr)=3445, 2953, 2868, 1731, 1698, 1257, 1029 cm⁻¹.

m.p.=142.2-144.4° C. (from EtOAc/hexanes mixture).

[α]_(D)=+92 (c=1% in CHCl₃).

ELSD Purity: 96.6%: Retention time=9.93 (Inertsil ODS 3V, 250×4.6 mm, 5um, ACN: 0.1% TFA in water (90:10)

Example 12

Methyl 3α-acetoxy-12α-hydroxy-5β-cholan-24-oate (2.1a)

A THF solution of lithium tri-tert-butoxyaluminum hydride (1 M, 22.4 mL,22.4 mmol) was added drop wise to a solution of compound 2.0a (2.5 g,5.6 mmol) in THF (25 mL) at ambient temperature. After stirring for anadditional 4-5 h, the reaction was determined to be complete by TLC. Thereaction was quenched by adding aqueous HCl (1 M, 10 mL) and the mixturewas diluted with EtOAc (30 mL). The phases were separated and theorganic phase was washed sequentially with water (15 mL) and saturatedbrine solution (10 mL). The organic phase was then dried over anhydrousNa₂SO₄ (3 g) and filtered. The filtrate was concentrated under vacuumand the resulting solid was purified by column chromatography [29 mm(W)×500 mm (L), 60-120 mesh silica, 50 g], eluting with EtOAc/hexane(2:8) [5 mL fractions, monitored by TLC with p-anisaldehyde charring].The fractions containing the product were combined and concentratedunder vacuum to provide compound 2.1a (2.3 g, 91%) as a white solid.

TLC: p-anisaldehyde charring, R_(f) for 2.1a=0.45 and R_(f) for2.0a=0.55.

TLC mobile phase: 30%-EtOAc in hexanes.

¹H NMR (500 MHz, CDCl₃): δ=4.68-4.73 (m, 1H), 3.98 (s, 1H), 3.66 (s,3H), 2.34-2.40 (m, 1H), 2.21-2.26 (m, 1H), 2.01 (s, 3H), 1.75-1.89 (m,6H), 1.39-1.68 (m, 16H), 1.00-1.38 (m, 3H), 0.96-0.97 (d, J=5.5 Hz, 3H),0.93 (s, 3H), 0.68 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ=174.5, 170.5, 74.1, 72.9, 51.3, 48.1, 47.2,46.4, 41.7, 35.8, 34.9, 34.7, 34.0, 33.5, 32.0, 30.9, 30.8, 28.6, 27.3,26.8, 26.3, 25.9, 23.4, 22.9, 21.3, 17.2, 12.6

Mass (m/z)=449.0 [M⁺+1], 466.0 [M⁺+18].

IR (KBr)=3621, 2938, 2866, 1742, 1730, 1262, 1162, 1041, cm⁻¹.

m.p=104.2-107.7° C. (from EtOAc).

[α]_(D)=+56 (c=1% in CHCl₃).

ELSD Purity: 97.0%: Retention time=12.75 (Inertsil ODS 3V, 250×4.6 mm, 5um. ACN:Water (60:40)

Example 13 Deoxycholic acid (DCA)

A solution of LiOH (187 mg, 4.4 mmol) in H₂O (2.0 mL) was added to asolution of compound 2.1a (500 mg, 1.11 mmol) in THF (8 mL) and MeOH (8mL). The resulting mixture was stirred for 3-4 h at 50° C. Upon completedisappearance of the starting material by TLC, the reaction mixture wasconcentrated under vacuum. A mixture of water (10 mL) and 3 N HCl (1 mL)were combined and cooled to 0° C. and then added to the crude product.After stirring for 1 h at 0° C., the precipitated solids were filteredand then washed with water (10 mL) and hexane (20 mL). Drying undervacuum at room temperature provided deoxycholic acid (DCA, 400 mg, 91%yield) as a white solid.

TLC: p-anisaldehyde charring, R_(f) for DCA=0.32 and R_(f) for2.1a=0.82.

TLC mobile phase: 10%-Methanol in DCM.

¹H NMR (500 MHz, DMSO): δ=11.92 (s, 1H), 4.44 (s, 1H), 4.19 (s, 1H),3.77 (s, 1H), 3.35-3.36 (m, 1H), 2.19-2.21 (m, 1H), 2.08-2.10 (m, 1H),1.73-1.80 (m, 4H), 1.43-1.63 (m, 6H), 1.15-1.35 (m, 12H), 0.98-1.05 (m,2H), 0.89-0.90 (d, J=6.0 Hz, 3H), 0.83 (s, 3H), 0.58 (s, 3H).

¹³C NMR (125 MHz, DMSO): δ=174.8, 71.0, 69.9, 47.4, 46.1, 46.0, 41.6,36.3, 35.6, 35.1, 34.9, 33.8, 32.9, 30.8, 30.7, 30.2, 28.6, 27.1, 27.0,26.1, 23.5, 23.0, 16.9, 12.4.

Mass (m/z)=393 [M⁺, +1].

IR=3363, 2933, 2863, 1694, 1453, 1372, 1042, cm⁻¹.

m.p.=171.4-173.6° C. (from ethanol); 174-176° C. (Alfa Aesar) and171-174° C. (Aldrich)

[α]_(D)=+47 (c=1% in EtOH), +54° (c=2% in ethanol) [Alfa Aesar]

ELSD Purity: 99.7%: Retention time=5.25 (Inertsil ODS 3V, 250×4.6 mm, 5um, ACN: 0.1% TFA in water (90:10).

Biological Example 1

Primary human adipocytes were incubated with varying concentrations ofsynthetic sodium deoxycholate synthesized using 9-HAD as startingmaterial or bovine-derived sodium deoxycholate obtained from Sigma asdescribed below.

Materials

Adipocytes (Zen-Bio cat# SA-1096)96 well plates (US Scientific cat# cellstar no. 655180)Serum-free RPMI medium (Mediatech cat#17-105-CV)Sodium deoxycholate (DC) (Sigma cat# D6750)Synthetic Sodium glycodeoxycholate (Kythera)

PBS (1×)

MTS assay kit (Promega cat# G3580)

Adipocytes arrived differentiated and at a density of 13,000 cells perwell in a 96 well plate. Two plates were received and each treated withthe same samples. Cells were incubated for 24 hours at 37° C. with 5%CO₂. A 1% stock solution of each bile acid (synthetic and non-syntheticDCA) were made by dissolving 20 mg into 2 mL media (serum-free). Usingthe 1% stock solution, the following 11 solutions were prepared bydilution: 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%,0.05%, 0.06%, and 0.1%, as well as 0% (media only).

Cells were washed 2× with 150 μL of room temperature 1×PBS (phosphatebuffered saline). Media and then PBS were removed from the wells in a 96well plate by turning the plate upside down and decanting the liquidinto a container. After the last PBS wash, 80 μL of sample was added perwell. Each concentration of a specific bile acid was added to 8 wellsand incubated for 1 hour at 37° C. with 5% CO₂. Plates were then removedfrom incubator and solution was decanted. A 100 μL solution of diluted(40 μL in 1 mL of RPMI) MTS reagent(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt) was added directly to each well. Plates were incubated at37° C. with 5% CO₂ until control (no bile acid) wells changed color toorange-brown and then loaded onto a spectrophotometer that analyzes 96well plates. Samples were run at 490 nm wavelength setting.

Cell viability was assessed using a colorimetric assay (MTS) kit fromPromega. The results show a dose-dependent decrease in cell survivalupon treatment with either syn-NaDC or Sigma-NaDC (see FIG. 1). Bothmolecules demonstrated similar cytolytic behavior in this experiment,indicating that synthetic-NaDC and bovine-derived Sigma-NaDC arefunctionally identical in terms of their ability to kill fat cells.

The embodiments and example described above are not intended to limitthe invention. It should be understood that numerous modifications andvariations are possible in accordance with the principles of the presentinvention.

1. A method of synthesis comprising

reacting a compound of formula 1.6 with an alkylpropiolate CH₂CH₂C(O)ORor an alkyl acrylate CH₂═CHC(O)OR wherein R is alkyl in the presence ofa Lewis acid to form a compound of formula 1.7 wherein P is a protectinggroup, R is a alkyl, and the dashed line

is a single or double bond, to provide a compound of formula 1.7

2-7. (canceled)
 8. The method of claim 1 wherein the Lewis acid of part(f) is EtCl₂.
 9. The method of claim 1 wherein the alkylpropiolate oralkylacrylate is methylpropriolate or methylacrylate. 10-12. (canceled)13. The method of claim 17 wherein the reducing agent of part (j) isLiAl(OtBu)₃H. 14-16. (canceled)
 17. A method synthesis comprisingreacting compound of formula 2.0

with a reducing agent to form a compound of formula 2.1 wherein P is aprotecting group and R is alkyl, to provide a compound of formula 2.1